1. Field of the Invention
The invention relates generally to the prostate and specifically to a novel complex of hK2 and PI-6 formed in the prostate, and methods of using the novel complex and its constituents.
2. Description of the Prior Art
Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the claims.
Three members of the human kallikrein family have been identified so far, designated hK1, hK2 and hK3 (1). All are serine proteases with high sequence identity. Two of these kallikreins, hK2 and hK3, are found almost exclusively in the prostate (extensively reviewed in (2). hK3, known more commonly as prostate-specific antigen, PSA, is a widely used serum marker for prostate. More recently, hK2 has become the focus of investigations into its possible role as a prostate cancer marker, as well as possible roles in prostate cancer biology (2).
hK2 is similar to PSA in many respects, such as prostate tissue localization (3; 4), 80% sequence identity (5; 6), and regulation by androgens (7; 8). From a biochemical perspective, hK2 is different from PSA in that it shows a strong trypsin-like activity, while PSA has weak chymotrypsin-like activity. However, unlike true kallikreins, hK2 shows little or no kininogenase-like activity (9; 10) and so does not appear to function primarily as a prostatically-expressed kininogenase.
The physiological roles for hK2 have not been established, though several activities have been described. hK2 has been shown to activate the zymogen form of PSA (pPSA) (11-13), and the zymogen of hK2 (autoactivation) (14). In this respect, hK2 is unique from the other human tissue kallikreins, hK1 and PSA. The activation of pPSA by hK2 is particularly interesting, since it suggests a possible physiological role for hK2 in the regulation of PSA activity.
Immunohistochemical studies using hK2-specific monoclonal antibodies have shown hK2 to be more highly expressed in prostate carcinoma than in normal tissues (15). This is the inverse of PSA, which tends to be lower in more poorly differentiated cancer epithelium than in normal tissues.
hK2 has been shown to activate urokinase-type plasminogen activator (uPA) by cleavage of the single-chain uPA at Lys158 to generate the two-chain active form of uPA (12; 16). It has been recently reported that hK2 rapidly complexes with PAI-1 in vitro and that hK2 also inactivates about 6 moles of PAI-1 during complex formation (17). It is therefore possible that the elevated levels of hK2 in prostate cancer play a biological role, either by the activation of urokinase or by the inactivation of PAI-1, the primary inhibitor of urokinase.
Physiologically, hK2 has been identified as a complex with ACT in serum (18) and PCI in seminal plasma (19). However, it was not previously known whether hK2 forms any tissue specific complex in the prostate. The physiological and biological role of hK2 in the prostate was also not known. Therefore, a need exists to study the role of hK2 in the prostate, particularly in prostate cancer. A need also exists to study the roles of any components that can form a complex with hK2.
The present invention is based on the discovery of a complex in prostate tissue extracts consisting of hK2 and a serine protease inhibitor known as protease inhibitor-6 (PI-6). It is also based on the discovery that the level of PI-6 is elevated in prostate tumors. Furthermore, it is the discovery of the present invention that different levels of PI-6 exist in different prostate carcinoma cells. Similar patterns were also observed in bladder cancer, kidney cancer and some breast cancer tumors. In addition, it is a discovery of the present invention that PI-6 is present in necrotic debris, but absent in prostate secretion products.
PI-6 was first reported in placental tissue, where it was called placental thrombin inhibitor due to its ability to form in vitro a complex with thrombin (20). It has also been called CAP, cytoplasmic antiprotease, since it has been shown to be cytoplasmically localized (21). PI-6 is expressed in epithelial and endothelial cells and has been described in a number of human tissues and cells including kidney, heart, skeletal muscle and platelets (22-24). It has not been reported in the prostate. In all cases so far, PI-6 appears to be cytoplasmically localized.
The discovery of the hK2-PI6 complex in the prostate is unique from the previous reports of hK2-ACT and hK2-PCI in at least two major respects: 1) PI-6 itself is a novel and relatively newly described member of the serine protease inhibitor family which has not been reported in prostate tissues; and 2) PI-6 is thought to be intracellular, which suggests either a novel pathway for the formation with the putatively extracellular hK2, or an altered pathway, possibly due to the oncogenic process.
The discovery that PI-6 exists in various amounts in cells of different physiological processes is also significant in that one may use PI-6 as a marker for diagnostic purposes, i.e., to identify prostate cancers and other cancers, or to distinguish different carcinoma cells of different physiological processes.
Accordingly, one aspect of the present invention provides a diagnostic method for determining the presence or absence of a condition selected from a group consisting of prostate cancer, bladder cancers, kidney cancers, breast cancers, and tissue necrosis. The method includes:
(a) contacting an amount of an agent, which specifically binds to PI-6, with a sample obtained from a human containing the PI-6 under a condition sufficient to allow the formation of a binary complex comprising the agent and the PI-6, and
(b) determining the amount of the complex in the sample and correlating the amount of the complex to the presence or absence of the condition in the human.
According to embodiments of the present invention, the sample may be a tissue sample, serum, seminal plasma, urine and blood. In one embodiment of the present invention, the sample is a prostate tissue sample, the condition is prostate cancer, and the agent comprises an antibody.
Another aspect of the present invention provides a diagnostic method for determining the physiological process of a cancer cell containing PI-6. The method includes:
(a) contacting an amount of an agent, which specifically binds to PI-6, with the cancer cell under a condition sufficient to allow the formation of a binary complex comprising the agent and the PI-6, and
(b) determining the amount of the complex in the cancer cell and correlating the amount of the complex to a physiological process of the cancer cell.
In accordance with embodiments of the present invention, the cancer cells may be prostate cancer cells, bladder cancer cells, kidney cancer cells, and breast cancer cells. The agent may comprise an antibody.
The invention is defined in its fullest scope in the appended claims and is described below in its preferred embodiments.